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Abstract

Background

Identifying novel tumor biomarkers to develop more effective diagnostic and therapeutic
strategies for patients with ACC is urgently needed. The aim of the study was to compare
the proteomic profiles between adrenocortical carcinomas (ACC) and normal adrenocortical
tissues in order to identify novel potential biomarkers for ACC.

Methods

The protein samples from 12 ACC tissues and their paired adjacent normal adrenocortical
tissues were profiled with two-dimensional electrophoresis; and differentially expressed
proteins were identified by mass spectrometry. Expression patterns of three differently
expressed proteins calreticulin, prohibitin and HSP60 in ACC, adrenocortical adenomas
(ACA) and normal adrenocortical tissues were further validated by immunohistochemistry.

Results

In our proteomic study, we identified 20 up-regulated and 9 down-regulated proteins
in ACC tissues compared with paired normal controls. Most of the up-regulated proteins
were focused in protein binding and oxidoreductase activity in Gene Ontology (GO)
molecular function classification. By immunohistochemistry, two biomarkers calreticulin
and prohibitin were validated to be overexpressed in ACC compared with adrenocortical
adenomas (ACA) and normal tissues, but also calreticulin overexpression was significantly
associated with tumor stages of ACC.

Conclusion

For the first time, calreticulin and prohibitin were identified to be novel candidate
biomarkers for ACC, and their roles during ACC carcinogenesis and clinical significance
deserves further investigation.

Keywords:

Background

Adrenocortical carcinomas (ACC) is an extremely rare malignancy, accounting for 0.2%
of cancer deaths annually in the world [1]. Most of ACC are carcinomatous, an extremely minor proportion of ACC tumors are characterized
by the presence sarcomatous components [2]. Recently, a couple of molecular pathways such as Wnt/β-catenin signaling have been
found to be involved in the carcinogenesis of ACC [3]. However, for the lack of early detection markers and effective treatments, ACC patients,
especially advanced-stage patients still have a poor prognosis [4].

Several recent studies have identified a panel of novel biomarkers or potential targets
for ACC. For example, Sbiera et al. [5] found that survivin overexpression was associated with a poor prognosis for ACC patients,
and targeting survivin might be an interesting new therapeutic approach for ACC. Gaujoux
S, et al. [6] confirmed that the presence of β-catenin nuclear staining is an independent prognostic
factor of overall and disease-free survival in patients with resected primary ACC.
Duregon et al. [7] suggest that detection of steroidogenic factor 1 (SF-1) could be a novel prognostic
marker in adrenocortical cancer. Insulin growth factor type 2 has been demonstrated
consistently overexpressed in ACC, and targeting its receptor IGF1R has shown encouraging
treatment potential [8]. However, candidate biomarkers for ACC are relatively limited compared with other
cancer types. Therefore, there is still an urgent need to identify novel tumor biomarkers
to develop more effective diagnostic and therapeutic strategies for patients with
ACC.

The comparative proteomic strategies provide useful tools in identifying novel biomarkers
for multiple cancers. According to our knowledge, until now, there is still no proteomic
study reported on ACC samples. In this study, we compare the proteomic profiles of
ACC tissues and their paired normal adrenocortical tissues by two-dimensional electrophoresis
(2-DE) and tandem mass spectrometry. A panel of proteins aberrantly expressed in ACC
tissues were identified, and part of them were further validated by immunohistochemistry
in a larger cohort of samples.

Materials and methods

Samples

For proteomic research, a total of 12 primary ACC tumor tissues and their paired adjacent
normal adrenocortical tissues were obtained from patients underwent resective operation
at Shandong Tumor Hospital, China. For it is hard to obtain normal healthy adrenocortical
tissues, we adopted normal adjacent adrenocortical tissues as a control of ACC in
our proteomic studies. Fresh ACC tissue (3–5 mm in diameter) were obtained from the
core part of cancer tissues without necrosis, and grossly normal adjacent tissues
were taken from the resection margin of ACC tumors. Resected fresh tissues were first
snap-frozen in liquid nitrogen, and stored at −80°C until use. For immunohistochemistry
validation study, a total of 39 ACC and paired normal adrenocortical tissues, and
31 benign adrenocortical adenomas (ACA) were also obtained from Shandong Tumor Hospital.
All the samples were histologically confirmed by two independent pathologists (D Mu
and De Zhang). The study was started upon approved by the ethical committee of our
institution, and samples were obtained with informed consent.

We adopted a “sample pool” strategy in the comparative proteomic study as described
previously [9]. Equal amount 500 μg of proteins extracted from ACC and normal adrenocortical tissues
were pooled respectively, and diluted with rehydration buffer (8 M urea, 2% CHAPS,
0.5% IPG buffer, 40 mM DTT) for isoelectric focusing. After isoelectric focusing,
the strips were first equilibrated with 130 mM DTT in equilibration buffer (6 M urea,
30% glycerol, 2% SDS, 50 mM Tris–HCl, pH 8.8), and then with 135 mM iodoacetamide
in the same buffer. SDS polyacrylamide (SDS-Page) was performed with constant power
(17 W/gel) at 20°C on an Ettan Dalt twelve system (GE healthcare). After the 2-DE,
the gels were stained with Coomassie blue R350 and images were scanned for data analysis
using Imagemaster 5.0 software package (GE healthcare).

In-gel digestion and mass spectrometry identification

The gel pieces were first destained with 25 mM NH4CO3/50% ACN for 30 min, and dehydrated in 100% ACN for 10 min, and were then digested
in 20 ng/μL sequencing grade-modified trypsin (Promega) overnight at 37°C. After extracted
with 5% TFA/50% ACN, the peptides were resuspended in 3 μL of 0.1% TFA for mass spectrometry
analysis. Protein identification was performed on 4700 Proteomic Analyzer MALDI-TOF-TOF
mass spectrometer (Applied Biosystems) in a reflective mode. All mass spectrometry
data were searched using the MASCOT search engine against a human subset of the Swiss-Prot
database.

Immunohistochemistry

Three proteins (calreticulin, prohibitin and HSP60) up-regulated in ACC identified
in the proteomic study were selected for validation in an independent set of samples
including 39 ACC, 31 ACA, and 39 normal adrenocortical tissues by immunohistochemistry
(IHC). Briefly, after rehydration and deparaffinization, paraffin-embedded tissue
slides were processed for antigen retrieval using heating in citrate buffer, and immunohistochemically
stained with the rabbit polyclonal antibodies against human calreticulin (1:150 dilution;
PTG-Lab) and prohibitin (1:150 dilution; PTG-Lab) and heat shock protein 60 (HSP60)
(1:100 dilution; PTG-Lab). All these antibodies were widely used in IHC staining,
and their specificity has been confirmed in many previous studies. Visualization was
performed using a SP kit (Maixin-Bio, Fujian, China). For the negative controls, the
primary antibody was replaced by rabbit IgG.

A semi-quantitative H score method was used to evaluate the results of IHC as described
previously within minor modification [10]. Staining intensity was quantified using the image analysis program Leica Qwin V3,
and were graded to four-scale (0–3), while the percentages of positive cells were
scored into four-scale (0, no positive cells; 1, 1%–33% positive cells; 2, 34%–66%;
and 3, 67%–100%). H score (ranging from 0 to 9) was calculated by multiplying staining
intensity and the percentage of positive cells. The median H-score was used as a cut-off
for classify low (< median H-score) and high (≥ median H-score) expression of each
markers.

Statistics

For proteomic study, spots with intensity changes greater than 2.0-fold were considered
as differently expressed spots, and were excised from gels for mass spectrometry analysis.
Gene Ontolgoy (GO) analysis was performed using MAS (Molecular Annotation System)
2.0 software (CapitalBio, China). For immunohistochemical results, the difference
in H scores of ACC, ACA, and normal controls were compared with Mann–Whitney t test, and the correlation between biomarkers with the clinicopathological traits
of ACC patients was evaluated with Chi-square or Student t test as appropriate. P value less than 0.05 was considered statistically significant.

Results

The 2-DE analyses were repeated in three replicas to guarantee the reproducibility
of the results. As seen in Figure 1, representative gel images were selected for comparative proteomic analyses of ACC
and their normal controls. According to the criteria established, spots with two fold
variation between the two groups were defined as differentially expressed proteins.
A total of 29 differentially expressed spots were successfully identified by mass
spectrometry. Twenty proteins were identified as being up-regulated in ACC samples,
compared with their corresponding proteins in normal adrenocortical tissues, while
9 proteins were identified to be down-regulated. The details of differently expressed
proteins were summarized in Table 1.

Expression of calreticulin, prohibitin and HSP60 in ACC, ACA and normal adrenocortical
tissues by immunohistochemistry

Three differentially expressed proteins, calreticulin, prohibitin and heat shock protein
60 (HSP60), which had not been reported in previous studies on ACC samples, were selected
to be validated in a larger size of samples by immunishotchemistry. As seen in Figure 2, the expression levels (H score) of calreticulin, prohibitin and HSP60 were significantly
higher in ACC samples than those in normal adrenocortical tissues, which are consistent
with the findings in proteomic study. Furthermore, ACC tumors also demonstrated a
higher expression level of calreticulin and prohibitin than ACA tumors, but the expression
of HSP60 showed no significant difference between malignant and benign adrenocortical
tumors. Negative staining with nonspecific rabbit IgG control was documented for each
experiment (data not shown).

Association of calreticulin and prohibitin expression with clinicopathological characteristics
in ACC

According to the criteria for IHC evaluation, the median H-score of 6 was set as the
cut-point to delineate low and high expression for calreticulin and prohibitin. The
relationship between calreticulin and prohibitin expression with clinicopathological
characteristics of ACC tumors was analyzed. As seen Table 3, no significant difference was observed between calreticulin and prohibitin expression
with all the chinicopathological characteristics of ACC tumors, except that calreticulin
overexpression was significantly associated with stages in ACC samples. High calreticulin
expression was seen more frequently in advanced-stage ACC tumors than in early-stage
cases (65.0% vs. 31.6%, P = 0.037).

Table 3.Association between calreticulin and prohibitin expression status with clinical charateristics
in ACC

Discussion

In this study, for the first time, we performed a 2-DE-based proteomic study to compare
the protein profiling of ACC and normal adrenocortical tissues. A panel of protein
markers were identified to be differently expressed. For only a few samples could
be included in traditional proteomic studies, we adopted a “sample pool” strategy
to increase the sample size. This strategy could also decrease the sample heterogeneity
in some extent. To validate the results of proteomic analysis, we further validate
three biomarkers calreticulin, prohibitin and HSP60 in a larger size of samples by
immunohistochemestry. These proteins were selected for the following reasons: first,
these biomarkers have a relatively high expression level in ACC, compared with normal
adrenocortical tissues; secondly, previous studies have indicated that these genes
are involved in the malignant progression of multiple cancers, but have not been evaluated
in ACC; third, commercial antibodies for immunohistochemistry are available.

Consistent with our proteomic findings, we confirmed calreticulin, prohibitin and
HSP60 overexpressed in ACC tumors than normal adrenocortical tissues. It has been
suggested that the protein profiling of benign tumors partly resemble their malignant
counterparts [11]. A candidate marker elevated in both ACC and ACA would lower their specificity in
ACC diagnosis. Therefore, we further compared the expression of calreticulin, prohibitin
and HSP60 in ACA and ACC. We found that HSP60 was overexpressed in both ACC and ACA,
compared with their normal controls, which would lower its further utility as a candidate
biomarker for ACC. Different from HSP60, ACC tissues had significantly higher expression
levels of calreticulin and prohibitin than ACA, supporting their utility as specific
biomarkers for ACC tumors.

Calreticulin was first identified as a Ca2+ binding protein, and has been implicated
in many cellular functions and pathophysiological process such as cell adhesion, autoimmunity
and heat shock [12,13]. Elevated expression of calreticulin has been reported in multiple cancers, and it
is proposed that the upregulation of calreticulin seems to be induced by cellular
stress from cancers [14]. Our results indicated that calreticulin correlated to tumor stage of ACC in clinical
samples. However, the exact mechanisms for its increases in ACC are as yet undetermined.

Prohibitin has been shown to localize to mitochondria, and has been identified to
be up-regulated in many cancers in previous studies [15]. However, experimental data about its role in tumorigenesis is conflicting. Several
studies have suggested that prohibitin effects as a tumor suppressor [16], while other data indicated that prohibitin is required for the activation of several
central signaling pathways related to carcinogenesis such as RAS-induced RAF-MEK-ERK
activation [17]. Our findings supported that prohibitin upregualted in ACC tumors and its roles in
ACC carcinogenesis deserves further investigation.

Except above three markers, we also identified other 17 up-regulated proteins in ACC,
most of which (such as transitional endoplasmic reticulum ATPase and 14-3-3 protein
epsilon) have been demonstrated to be involved in cancer carcinogenesis in other cancers
in previous studies [18-20], but have not been reported to be associated with ACC. Therefore, these proteins
might also be novel potential candidate markers for ACC, and deserves further investigation
in the future.

Conclusions

In this proteomic study, we identified and validated calreticulin and prohibitin overexpressed
in ACC samples compared with their normal and benign counterparts, suggesting that
these two markers are novel potential candidate biomarkers for ACCs. We proposed that
the molecular mechanisms of calreticulin and prohibitin during ACC carcinogenesis
also deserve further investigation in the future.

Competing interests

The authors have declared no conflicts of interest.

Authors’ contribution

YMS designed the study, performed the experiments, analyzed the data and drafted the
manuscript; ZB, ZX, SXT, SXW, XS were responsible for the samples collection; WH and
WB participated in proteomic studies; LW, PZ, GY participated in IHC; MD and ZD were
responsible for the histological examination of specimens and IHC results evaluation
and score. All the authors read and approved the final manuscript.

Acknowledgements

This study is supported by the project of Shandong provincial doctor fund (2008BS02017).